Skip to main content
SpringerLink
Log in

Cloning and characterization of a trypsin inhibitor cDNA from amaranth (Amaranthus hypochondriacus) seeds

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

We previously isolated and sequenced the major trypsin inhibitor from Amaranthus hypochondriacus seeds. This amaranth trypsin inhibitor (AmTI) is a 69 amino acid protein with high homology to members of the potato-1 inhibitor family. This paper describes the cloning and expression of a cDNA encoding this trypsin inhibitor in various vegetative tissues of the amaranth plant during seed development and imbibition, and investigates the possible induction of AmTI expression by wounding.

We obtained a 393 bp cDNA sequence with an open reading frame corresponding to a polypeptide with 76 amino acid residues. With the exception of one residue (Ser-41), the polypeptide agrees with the amino acid sequence previously reported, plus 7 more residues at the N-terminus. These N-terminal residues are thought to be part of the signal used for intracellular sorting.

The organ specificity of AmTI gene expression was investigated by northern analysis, showing that mRNA corresponding to AmTI genes was present in stems of plants growing under normal conditions.

The kinetics of accumulation of the AmTI-mRNA, protein, and inhibitory activity during seed development and imbibition was determined. AmTI-mRNA accumulation reached a maximum at 14 days after anthesis (daa) and then gradually decreased, being barely detectable 36 daa. The AmTI protein accumulation followed the same profile as the inhibitory activity, both were delayed with respect to the mRNA. The maximum level was observed 22 daa, and then gradually decreased until a steady state was reached as seed maturation proceeded. Upon imbibition, a gradual decrease in AmTI protein and inhibitory activity was shown; however, an AmTI transcript was detected 24 h after imbibition. In contrast to representative members of the potato I family, this inhibitor was not inducible by wounding of leaves.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Altpeter, F., Díaz, I., McAuslane, H., Gaddour, K., Carbonero, P. and Vasil, I.K. 1999. Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe. Mol. Breed. 5: 53-63.

    Google Scholar 

  • Barba de la Rosa, A.P., Herrera-Estrella, A., Utsumi, S. and Paredes-López, O. 1996. Molecular characterization, cloning and structural analysis of a cDNA encoding an amaranth globulin. J. Plant Physiol. 149: 527-532.

    Google Scholar 

  • Baumgartner, B. and Chrispeels, M.J. 1997. Purification and characterization of vicilin peptidohydrolase, the major endopeptidase in the cotyledons of mung-bean seedlings. Eur. J. Biochem. 77: 223-233.

    Google Scholar 

  • Bednarek, S.Y. and Raikel, N.V. 1992. Intracellular trafficking of secretory proteins. Plant Mol. Biol. 20: 133-150.

    Google Scholar 

  • Belozersky, M.A., Dunaevsky, Y.E., Musolyamov, A.X. and Egorov, T.A. 1995. Complete amino acid sequence of the protease inhibitor from buckwheat seeds. FEBS Lett. 371: 264-266.

    Google Scholar 

  • Beuning, L.L., Spiiggs, T.W. and Christeller, J.T. 1994. Evolution of the proteinase inhibitor I family and apparent lack of hypervariability in the proteinase contact loop. J. Mol. Evol. 39: 644-654.

    Google Scholar 

  • Boller, T. and Kende, H. 1979. Hydrolytic enzymes in the central vacuole of plant cell. Plant Physiol. 63: 1123-1132.

    Google Scholar 

  • Boulter, D. 1993. Insect pest control by copying nature using genetically engineered crops. Phytochemistry 34: 1453-1466.

    Google Scholar 

  • Bressani, R. 1994. Composition and nutritional properties of amaranth. In: O. Paredes-López (Ed.), Amaranth Biology, Chemistry, and Technology, CRC Press, New York, pp. 185-205.

    Google Scholar 

  • Chagolla-López, A., Blanco-Labra, A., Patthy, A., Sánchez, R. and Pongor, S. 1994. A novel _-amylase inhibitor from amaranth (Amaranthus hypochondriacus) seeds. J. Biol. Chem. 269: 23675-23680.

    Google Scholar 

  • Goldberg, R.B., Barker, S.J. and Perez-Grau, L. 1989. Regulation of gene expression during plant embryogenesis. Cell 56: 149-160.

    Google Scholar 

  • Habu, Y., Fukushima, H., Sakata, Y., Abe, H. and Funada, R. 1996. A gene encoding a major Kunitz proteinase inhibitor of storage 23 organs of winged bean is also expressed in the phloem of stems. Plant Mol. Biol. 32: 1209-1213.

    Google Scholar 

  • Hara-Nishimura, I., Shimada, T., Hiraiwa, N. and Nishimura, M. 1995. Vacuolar processing enzyme responsible for maturation of seed proteins. J. Plant Physiol. 145: 632-640.

    Google Scholar 

  • Heigaard, J., Dam, J., Petersen, L.C. and Bjorn, S.E. 1994. Primary structure and specificity of the major serine proteinase inhibitor of amaranth (Amaranthus caudatus L.) seeds. Biochim. Biophys. Acta 1204: 68-74.

    Google Scholar 

  • Hernández, S., Sánchez, M., Guzmán, P. and Simpson, J. 1995. Isolation of high molecular weight DNA from plant nuclei. Meth. Mol. Cell. Biol. 5: 349-352.

    Google Scholar 

  • Jofuku, K.D. and Goldberg, R.B. 1989. Kunitz trypsin inhibitor genes are differentially expressed during the soybean life cycle and in transformed tobacco plants. Plant Cell 1: 1079-1093.

    Google Scholar 

  • Kai-Wun, Y., Jen-Chih, Ch., Mei-In, L., Yih-Ming, Ch. and Chu-Yung, L. 1997. Functional activity of sporamin from sweet potato (Ipomoea batatas Lam.): a tuber storage protein with trypsin inhibitory activity. Plant Mol. Biol. 33: 565-570.

    Google Scholar 

  • Kozak, M. 1991. Structural features in eukaryotic mRNA that modulate the initiation of translation. J. Biol. Chem. 266: 19867-19870.

    Google Scholar 

  • Krishnamoorty, R., Gong, Y. and Richardson, M. 1990. A new protein inhibitor of trypsin activated Hageman factor from pumpkin (Cucurbita maxima) seeds. FEBS Lett. 273: 163-167.

    Google Scholar 

  • Kyte, J. and Doolittle, R.F. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105-132.

    Google Scholar 

  • Muntz, K. 1998. Deposition of storage proteins. Plant Mol. Biol. 38: 77-99.

    Google Scholar 

  • Richardson, M. 1991. Seed storage proteins: the enzyme inhibitors. In: L. Rogers (Ed.), Methods in Plant Biochemistry, Academic Press, New York, pp. 259-305.

    Google Scholar 

  • Ryan, C.A. 1990. Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu. Rev. Phytopathol. 28: 425-449.

    Google Scholar 

  • Sambrook J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  • Sanger, F. 1977. DNA sequencing with chain termination inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.

    Google Scholar 

  • Schägger, H. and von Jagow, G. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of protein in the range from 1 to 100 kDa. Anal. Biochem. 166: 368-379.

    Google Scholar 

  • Schuler, M.A. and Zielinski, R.E. 1989. RNA isolation from light and dark grown seedlings. In: M.A. Schuler (Ed.), Methods in Plant Molecular Biology, Academic Press, San Diego, CA, pp. 89-96.

    Google Scholar 

  • Schwert, G.W. and Takenaka, Y. 1955. A spectrophotometric determination of trypsin and chymotrypsin activity. Biochim. Biophys. Acta 16: 571-575.

    Google Scholar 

  • Segura-Nieto, M., Vázquez-Sánchez, N., Rubio-Velázquez, H., Olguín-Martínez, L., Rodríguez-Nester, C. and Herrera-Estrella, L. 1992. Chacterization of amaranth (Amaranthus hipochondriacus) seed proteins. J. Agric. Food Chem. 40: 1553.

    Google Scholar 

  • Stemmer, W.P. 1991. A 20-minute ethidium bromide/high-salt extraction protocol for plasmid DNA. Biotechniques 10: 726.

    Google Scholar 

  • Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA 73: 4350-4354.

    Google Scholar 

  • Valdés-Rodríguez, S., Segura-Nieto, M., Chagolla-López, A., Verver y Vargas-Cortina, A., Martínez-Gallardo, N. and Blanco-Labra, A. 1993. Purification, characterization, and complete amino acid sequence of a trypsin inhibitor from amaranth (Amaranthus hypochondriacus) seeds. Plant Physiol. 103: 1407-1412.

    Google Scholar 

  • Wilson, K. 1980. The release of proteinase inhibitors from legume seeds during seed germination. Phytochemistry 19: 2517-2519.

    Google Scholar 

  • Wingate, V. and Ryan, C. 1991. A novel fruit-expressed trypsin inhibitor I gene from a wild species of tomato. J. Biol. Chem. 266: 5184-5188.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Centro de Investigación y de Estudios Avanzados del IPN, Unidad de Biotecnología e Ingeniería Genética de Plantas, Apdo postal 629, C.P. 36500, Irapuato, Guanajuato, México

    Silvia Valdés-Rodríguez, Alejandro Blanco-Labra, Glenda Gutiérrez-Benicio, Alfredo Herrera-Estrella & June Simpson

  2. Instituto de Ciencias Agrícolas, Universidad de Guanajuato, Apdo postal 311, C.P. 36500 and, Irapuato, Guanajuato, México

    Anatoli Boradenenko

Authors
  1. Silvia Valdés-Rodríguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alejandro Blanco-Labra
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Glenda Gutiérrez-Benicio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anatoli Boradenenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alfredo Herrera-Estrella
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. June Simpson
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Valdés-Rodríguez, S., Blanco-Labra, A., Gutiérrez-Benicio, G. et al. Cloning and characterization of a trypsin inhibitor cDNA from amaranth (Amaranthus hypochondriacus) seeds. Plant Mol Biol 41, 15–23 (1999). https://doi.org/10.1023/A:1006262106267

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1006262106267

Search

Navigation